Two photon excitation offers significant advantages over other types of microscopy-based imaging, especially for imaging living cells either in vivo or in thick slices. Indeed, the technology underlying two photon imaging has rapidly advanced over the last few years. The objective of this proposal is to provide access to this technology to the growing MIT Neuroscience community. This instrument will enable researchers to perform simultaneous imaging of two or more fluorescent proteins in experiments combining scanning with sophisticated techniques, including electrophysiological recording, stimulation of cells containing channelrhodopsins or halorhodopsins, or rapid uncaging of bioactive molecules. Access to these cutting-edge techniques will enhance a broad variety of research programs, and facilitate an expansive array of NIH-funded research, including studies addressing the following: molecular regulation of synapse formation in the developing brain, factors controlling the migration and integration of newly born neurons in the adult brain, neurite regeneration following injury in C. elegans, structural changes following fear learning and extinction, factors governing inhibitory synapse formation and stabilization, the role of protein misfolding in the progression of mouse models of Parkinson's and prion disease, factors regulating the development of brain ventricles, studies of the role of inhibitory interneurons and astrocytes in visual cortex dynamics, spine dynamics in mouse models of autism and schizophrenia, and the contribution of postsynaptic scaffolding proteins to synaptic plasticity. This instrument will also be used to further the advancement of two photon imaging technologies, by enabling research aimed at developing software scanning protocols for rapid two-photon activation of cells and rapid readout of neural activity, and particularly facilitating the development of algorithms for rapid identification of cells during the imaging process.